CN106137968A

CN106137968A - Inner membrance reversible crosslink Biodegradable polymer vesicles with positive electricity and preparation method thereof and the application in preparing antitumor drug

Info

Publication number: CN106137968A
Application number: CN201610558116.6A
Authority: CN
Inventors: 孟凤华; 方媛; 杨炜静; 邹艳; 钟志远
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2016-07-15
Filing date: 2016-07-15
Publication date: 2016-11-23
Anticipated expiration: 2036-07-15
Also published as: CN108542885B; CN108542885A; WO2018010624A1; CN106137968B

Abstract

The present invention sets and discloses inner membrance reversible crosslink Biodegradable polymer vesicles with positive electricity and preparation method thereof and the application in preparing antitumor drug；Cancer target based on block polymer PEG P (TMC DTC) SP or PEG P (LA DTC) SP, there is positively charged inner membrance, reduction sensitive reversible crosslink, the intracellular Biodegradable polymer vesicles solving crosslinking; can efficiently load protection biomacromolecule such as protein, DNA and siRNA and the electronegative small-molecule drug of physiological environment; and in the defeated tumor cell sending them to live body of energy, induce its apoptosis.This system has multiple particular advantages, including the most handling, outstanding biocompatibility, control release property, superpower body-internal-circulation stability, superior cancerous cell targeting, the significant cancer cell-apoptosis ability etc. fabulous to medicine of preparation.Therefore, it is expected to become the nanosystems platform integrating the advantage such as simple, stable, multi-functional, for efficiently, active targeting conveying nucleic acid is to tumor in situ.

Description

Inner membrance has reversible crosslink Biodegradable polymer vesicles and the preparation side thereof of positive electricity Method and the application in preparing antitumor drug

Technical field

The invention belongs to technical field of pharmaceuticals, be specifically related to a kind of inner membrance and there is the reversible crosslink biodegradable poly of positive electricity Compound vesicle and preparation method thereof and the application in the conveying of the electronegative small-molecule drug of bio-pharmaceutical and physiological environment.

Background technology

Cancer is to threaten the primary killers of human health, and its M & M is in the trend risen year by year.By parents Property the polymer vesicle performance schedulable that formed of polymer self assembles big, hydrophilic medicament and hydrophobic drug can be loaded simultaneously, It it is the ideal carrier preparing anti-tumor nano medicine.The especially water quality inner chamber of vesicle can be biopharmaceutical macromolecular drug such as albumen medicine Thing and nucleic acid drug provide ideal space.But existing vesicle is to the little biopharmaceutical macromolecular drug of these toxic and side effects and life The efficiency of loading of the reason electronegative small molecule anticancer drug of environment is low, significantly limit its answering in these pharmaceutical preparatioies With.

At present, although gene therapy can be transported to organism specific tissue cell the gene with specific function Treatment disease, siRNA s (siRNA) the most in recent years includes for treatment as a kind of novel nucleic acids medicine especially Cancer in interior incurable disease, is different from the small-molecular-weight of DNA, has only in Cytoplasm, need not enter nucleus and play Effect, has huge application potential.But these genomic medicines are easily by nuclease degradation, enter cell ability poor, non-specific Property is missed the target, immunogenicity is high all hinders its clinical practice.Although the carrier transfection efficiency with virus as nucleic acid drug is the highest, But safety causes anxiety, there is high immunogenicity and potential carcinogenecity.Therefore non-viral gene vector especially cationic polymerization Thing genophore becomes the focus of research, loads nucleic acid with nano-carriers such as the liposome of cation and poly ion complexes Result of study also and unsatisfactory, there is internal instability, targeting is poor, gene is compound and transfection efficiency is the highest or The problem that cytotoxicity is high.There is no the scheme that can simultaneously solve these problems at present.

The active height of pharmaceutical grade protein, high specificity, side effect are little, not by the advantages such as cellular drug resistance is affected, right and wrong Often there is development potentiality as new type antineoplastic medicine.But protein volume the most easily enter intracellular, be prone to be dropped by protease Solve, be prone to degeneration.Pemetrexed disodium is containing the folic acid resisting preparation that core is pyrrolopyrimidine group in a kind of structure, by broken The dependent normal metabolic processes of folic acid in bad cell, suppresses cellular replication, thus suppresses the growth of tumor.Clinical medicine is note Penetrate liquid, the malignant pleural mesothelioma cannot performed the operation for treatment and the Second line Drug of nonsmall-cell lung cancer.But especially physiologic ring The electronegative inefficient causing its entrance cells play effect under border.

But in the polymer vesicle technology of existing preparation, still lack stable circulation, selectively targeted tumor, thin in vivo The efficient nano vesicle that intracellular rapid delivery of pharmaceuticals, toxic and side effects are little, especially lack to efficiency of loading high, play guarantor very well Protect the polymer vesicle that the biocompatibility of effect is excellent.

Summary of the invention

It is an object of the invention to disclose a kind of inner membrance have positive electricity reversible crosslink Biodegradable polymer vesicles and Preparation method.

To achieve the above object of the invention, the present invention adopts the following technical scheme that

A kind of inner membrance has the reversible crosslink Biodegradable polymer vesicles of positive electricity, crosslinking after polymer self assembles obtain； The strand of described polymer includes hydrophilic segment, hydrophobic segment and the spermine molecule being sequentially connected with；Described hydrophobic segment bag Include Merlon segment and/or polyester segment；The molecular weight of described hydrophilic segment is 2000-8000Da；The molecule of hydrophobic segment Amount is 2.3-8.4 times of hydrophilic segment molecular weight.

Preferably, the polymeric chemical structure formula of the present invention is as follows:

Wherein, R₁One in following group:

R₂One in following group:

、；

In described polymer, the molecular weight of PEG is 3000-10000Da；The total molecular weight of PTMC or PLA is the 2-of PEG molecular weight 6 times；The total molecular weight of PDTC is the 15%-40% of PTMC or PLA total molecular weight.

In the polymer of the present invention, spermine is little as toxicity during carrier, in conjunction with PEG chain segment and hydrophobic segment, can be formed Good drug encapsulation effect, even if when siRNA content is up to 80wt .%, this vesicle still can completely, consolidation parcel SiRNA, and can efficiently load protein such as cytochrome C；The polymer of the present invention avoids existing cationic polymerization simultaneously Instability that objects system bind nucleic acid by the way of physical entanglement brings, positively charged easily with Cell binding and migration force is poor, release Put inefficient defect；The present invention by electrostatic force composite nucleic acid, protein or electronegative small-molecule drug, then by The vesicle film of crosslinking and extraneous separation, it is to avoid caused damage and toxic and side effects by cell adhesion at course of conveying, it is possible to efficiently Deliver to affected area, and under the effect of high salt concentration and reducing agent GSH in vivo, quickly discharge nucleic acid drug, solve disease problems.

In the present invention, polymer vesicle is that inner membrance has the reduction sensitivity reversible crosslink of positive charge, intracellular solves crosslinking Biodegradable polymer vesicles；Described polymer is PEG-P (TMC-DTC)-SP or PEG-P (LA-DTC)-SP, the most poly- Compound is by PEG hydrophilic segment, hydrophobic segment and spermine molecular composition, and wherein the structure of hydrophobic segment is:

；

Work as R₂ForTime, for PTMC segment；Work as R₂ForTime, for PLA segment, the most hydrophobic Segment is made up of P (TMC-co-DTC) or P (LA-co-DTC).

Preferred version is: PEG molecular weight is 5000-7500Da；The total molecular weight of PTMC or PLA is PEG molecular weight 2.5-5 again；The total molecular weight of PDTC is the 18%-38% of PTMC or PLA total molecular weight.

Spermine, the reversible crosslink biodegradable polymer capsule based on inner membrance with positive charge of present invention design Bubble, it is possible to realize the efficient loading to biopharmaceutical macromolecular drug and electronegative small molecule anticancer drug.Spermine contains two ammonia Base and two imino groups, be present in antibacterial and most animals cell, is the important substance promoting cell proliferation.Above-mentioned three embedding Section polymer P EG-P (TMC-DTC)-SP or PEG-P (LA-DTC)-SP, wherein TMC or LA and DTC of mid-block in Random arrangement；The molecular weight of spermine is 202Da, much smaller than PEG molecular weight, obtains inner membrance and have positive electricity after self assembly, crosslinking The polymer vesicle of the crosslinking of lotus, the inner shell of vesicle film is that spermine is for compound bio macromole such as protein, DNA and siRNA Small-molecule drug electronegative with physiological environment；Vesicle film is biodegradable and the PTMC of good biocompatibility of reversible crosslink Or PLA, the dithiolane of side chain is similar to the antioxidant thioctic acid that human body is natural, it is possible to provide the reversible crosslink that reduction is sensitive, Not only biological support medicine long circulating in blood, it is also ensured that cross-link in intracellular quick solution, release medicine is to target cell Intracellular.

The invention also discloses the preparation side that above-mentioned inner membrance has the reversible crosslink Biodegradable polymer vesicles of positive electricity Method, comprises the following steps:

(1) by the end of PEG-P (TMC-DTC) or PEG-P (LA-DTC) hydroxy activating reagent such as chloro-carbonic acid p-nitrophenyl Ester NPC activates, then reacts prepared PEG-P (TMC-DTC)-SP or PEG-P (LA-DTC)-SP with spermine；

(2) at the PEG end coupling tumour-specific targeted molecular of PEG-P (TMC-DTC)-SP or PEG-P (LA-DTC) SP, Obtain targeting PEG-P (TMC-DTC)-SP or targeting PEG-P (LA-DTC)-SP；

(3) with PEG-P (TMC-DTC)-SP or PEG-P (LA-DTC)-SP as raw material, prepare inner membrance by solvent displacement and have There is the reversible crosslink Biodegradable polymer vesicles of positive electricity；Or with PEG-P (TMC-DTC)-SP and targeting PEG-P (TMC- DTC)-SP is raw material, prepare cancer target by solvent displacement, inner membrance have positive electricity reversible crosslink biodegradable polymerization Thing vesicle；Or, prepared by solvent displacement as raw material with PEG-P (LA-DTC)-SP and targeting PEG-P (LA-DTC)-SP Cancer target, inner membrance have the reversible crosslink Biodegradable polymer vesicles of positive electricity；Or with PEG-P (TMC-DTC)-SP and Targeting PEG-P (TMC-DTC) is raw material, prepares inner membrance by solvent displacement and has the reversible crosslink biodegradable poly of positive electricity Compound vesicle；Or with PEG-P (LA-DTC)-SP and targeting PEG-P (TMC-DTC) as raw material, prepared by solvent displacement Inner membrance has the reversible crosslink Biodegradable polymer vesicles of positive electricity.

Preferably with PEG-P (TMC-DTC)-SP and targeting PEG-P (TMC-DTC) as raw material, or with PEG-P (LA- DTC)-SP and targeting PEG-P (LA-DTC) is that raw material is blended, and self assembly, crosslinking obtains tumor-targeting, inner membrance has positive electricity The polymer vesicle of lotus, shell be with PEG as background, targeted molecular to cancerous cell can high specific combine, increase carrier target Tropism.Targeted molecular can be polypeptide DP8, cNGQ, cRGD, CC9, folic acid FA or galactose Gal.Such as by PEG-P (TMC- DTC)-SP or PEG-P (LA-DTC)-SP and the coupling diblock polymer such as DP8-PEG-P of tumor-targeting molecule (TMC-DTC) mixing, altogether self assembly, load medicine, crosslinking after obtain tumor-targeting, inner membrance has the polymer of positive charge Vesicle (DP8-RCCPs)；The chemical structural formula of described DP8-PEG-P (TMC-DTC) is:

。

Above-mentioned preparation method, specifically includes following steps:

Step (1) is by PEG-P (TMC-DTC) or PEG-P (LA-DTC), hydroxy activating reagent p-nitrophenyl chloroformate ester NPC Be dissolved in dry solvent reaction, then precipitate, filter, be vacuum dried obtain activation PEG-P (TMC-DTC)-NPC or PEG-P(LA-DTC)-NPC；PEG-P (TMC-DTC)-NPC or PEG-P (LA-DTC)-NPC solution is added drop-wise to solution of spermine After middle reaction, dialyse, precipitate, sucking filtration, vacuum drying obtain PEG-P (TMC-DTC)-SP or PEG-P (LA-DTC)-SP；Step Suddenly (2) are for be dissolved in obtaining polymer in the organic solvent with targeted molecular such as DMSO or DMF；Step (3) is that raw material is molten Liquid adds in nonionic buffer solution such as HEPES, room temperature place a little after identical buffer solution is dialysed, hatch crosslinking, must There is the reversible crosslink Biodegradable polymer vesicles of positive electricity to inner membrance.The present invention adding or can be not added with reducing agent such as two sulfur For the inner membrance that obtains normal temperature crosslinked under threitol (DTT) and glutathion (GSH), there is the reversible crosslink biodegradable poly of positive electricity Compound vesicle.

Such as:

By the terminal hydroxyl of PEG-P (TMC-DTC) with hydroxy activating reagent such as p-nitrophenyl chloroformate ester (NPC) activate, then with essence The end group primary amine reaction of amine prepares PEG-P (TMC-DTC)-SP.Specifically, PEG-P (TMC-DTC) and NPC is dissolved in dry two Chloromethanes (DCM) reacts 12-24 hour under ice-water bath, then precipitates in ice ether, filter, be vacuum dried and obtain PEG-P (TMC-DTC)-NPC；Then PEG-P (TMC-DTC)-NPC is dissolved in dry DCM, is added drop-wise in the DCM of spermine at 30-40 DEG C After reacting 12-24 hour, dialysis 24-48 hour in DCM and methanol (volume ratio is 1:1), then precipitation, sucking filtration, vacuum are done Dry obtain product PEG-P (TMC-DTC)-SP；

With PEG-P (TMC-DTC)-SP and targeting PEG-P (TMC-DTC) as raw material, prepare inner membrance by solvent displacement and have The self-crosslinking polymer vesicle of positive charge；It is specially PEG-P (TMC-DTC)-SP and the DMSO of targeting PEG-P (TMC-DTC) Add in HEPES buffer after solution mixing, kept at room temperature overnight, dialysis, adding or be not added with reducing agent such as dithio threose Hatch 4 h vesicle crosslinkings when alcohol (DTT) or glutathion (GSH), obtain inner membrance and there is the cross linked polymer vesicle of positive charge.

The present invention further discloses a kind of antitumor drug, above-mentioned inner membrance the reversible crosslink biology with positive electricity can drop Depolymerization compound vesicle loads medicine and obtains；Described medicine is the electronegative small-molecule drug of protein, nucleic acid or physiological environment, as Pemetrexed, methotrexate.The medicine of the present invention with active targeting, the sensitive reversible crosslink of reduction nano vesicle as carrier, dress Carrying anti-tumor medicine, curative effect remarkable at Mice Body internal therapy Tumors display and hypotoxicity.

The preparation method of above-mentioned antitumor drug, the one in following preparation method:

(1) PEG-P (TMC-DTC)-SP solution or PEG-P (LA-DTC)-SP solution are mixed with medicine, nonionic buffer, Room temperature is placed, and then dialyses, hatches crosslinking and obtain antitumor drug；

(2) PEG-P (TMC-DTC)-SP solution and targeting PEG-P (TMC-DTC)-SP solution are delayed with drug solution, nonionic Rushing liquid mixing, room temperature is placed, and then dialyses, hatches crosslinking and obtain antitumor drug；

(3) PEG-P (LA-DTC)-SP solution and targeting PEG-P (LA-DTC)-SP solution, medicine, nonionic buffer solution are mixed Closing, room temperature is placed, and then dialyses, hatches crosslinking and obtain antitumor drug；

(4) PEG-P (TMC-DTC)-SP solution and targeting PEG-P (TMC-DTC) solution are mixed with medicine, nonionic buffer solution Closing, room temperature is placed, and then dialyses, hatches crosslinking and obtain antitumor drug；

(5) PEG-P (LA-DTC)-SP solution and targeting PEG-P (LA-DTC) solution are mixed with medicine, nonionic buffer solution Closing, room temperature is placed, and then dialyses, hatches crosslinking and obtain antitumor drug.

Wherein, polymer solution, antineoplastic drug solution and nonionic buffer solution three are mixed to get bag medicine carrying jointly The inner membrance of thing has the reversible crosslink Biodegradable polymer vesicles of positive electricity, and optimum condition is that polymer solution adds to containing egg In the HEPES buffer solution of the electronegative small-molecule drug of white matter, DNA and physiological environment, or polymer solution and siRNA solution Add after mixing in HEPES, such as:

By protein or DNA or the electronegative small-molecule drug of physiological environment, nonionic buffer such as HEPES mixing, add PEG-P (TMC-DTC)-SP solution or PEG-P (LA-DTC)-SP solution, room temperature is placed, and then dialyses, hatches crosslinking and obtain Antitumor drug；

By protein or DNA or the electronegative small-molecule drug of physiological environment, nonionic buffer such as HEPES mixing, add PEG-P (TMC-DTC)-SP solution and targeting PEG-P (TMC-DTC)-SP solution, room temperature is placed, and then dialyses, hatches and cross-link To antitumor drug；

Protein or DNA or the electronegative small-molecule drug of physiological environment, nonionic are delayed solution such as HEPES mixing, adds PEG-P (LA-DTC)-SP solution and targeting PEG-P (LA-DTC)-SP solution, room temperature is placed, and then dialyses, hatches crosslinking and obtain Antitumor drug；

By protein or DNA or the electronegative small-molecule drug of physiological environment, nonionic buffer such as HEPES mixing, add PEG-P (TMC-DTC)-SP solution and targeting PEG-P (TMC-DTC) solution, room temperature is placed, and then dialyses, hatches crosslinking and obtain Antitumor drug；

By protein or DNA or the electronegative small-molecule drug of physiological environment, nonionic buffer such as HEPES mixing, add PEG-P (LA-DTC)-SP solution and targeting PEG-P (LA-DTC) solution, room temperature is placed, and then dialyses, hatches crosslinking and resisted Tumour medicine.

Preferably, PEG-P (TMC-DTC)-SP solution or PEG-P (LA-DTC)-SP solution are mixed with siRNA solution Close, add in nonionic buffer such as HEPES, room temperature placement, then dialyse, hatch crosslinking and obtain antitumor drug；

PEG-P (TMC-DTC)-SP solution and targeting PEG-P (TMC-DTC)-SP solution are mixed with siRNA solution, adds As in HEPES in nonionic buffer, room temperature is placed, and then dialyses, hatches crosslinking and obtain antitumor drug；

PEG-P (LA-DTC)-SP solution and targeting PEG-P (LA-DTC)-SP solution are mixed with siRNA solution, adds non- In ion buffer such as HEPES, room temperature is placed, and then dialyses, hatches crosslinking and obtain antitumor drug；

PEG-P (TMC-DTC)-SP solution and targeting PEG-P (TMC-DTC) solution are mixed with siRNA solution, add non-from As in HEPES in sub-buffer, room temperature is placed, and then dialyses, hatches crosslinking and obtain antitumor drug；

PEG-P (LA-DTC)-SP solution and targeting PEG-P (LA-DTC) solution are mixed with siRNA solution, adds nonionic As in HEPES in buffer, room temperature is placed, and then dialyses, hatches crosslinking and obtain antitumor drug.

The invention also discloses above-mentioned inner membrance and there is the reversible crosslink Biodegradable polymer vesicles of positive electricity as anti-swollen The application of tumor nano-medicament carrier, for example as protein, siRNA, DNA and the physiological environment band such as pemetrexed, methotrexate The application of the carrier of the small-molecule drug of negative electricity.

The invention also discloses above-mentioned inner membrance and there is the reversible crosslink Biodegradable polymer vesicles of positive electricity in preparation life Application in thing antitumor drug.

The invention also discloses a kind of polymer, it is characterised in that the chemical structural formula of described polymer is as follows:

Wherein, R₁One in following group:

R₂One in following group:

、；

In described polymer, the molecular weight of PEG is 2000-8000Da；The total molecular weight of PTMC or PLA is the 2-6 of PEG molecular weight Times；The total molecular weight of PDTC is the 15%-40% of PTMC or PLA total molecular weight.

Compared with prior art, present invention have the advantage that

1. the present invention devises inner membrance and has the cross linked polymer vesicle internal transmission for antitumor drug of positive charge；First First having synthesized block polymer PEG-P (TMC-DTC)-SP, spermine is owing to molecular weight is much smaller than PEG molecular weight, at polymer certainly Obtaining the cross linked polymer vesicle that inner shell is spermine of vesicle film after assembling, crosslinking, the spermine of vesicle film inner shell is for efficiently dress Carry protein, DNA, siRNA and the electronegative small-molecule drug of physiological environment such as pemetrexed, methotrexate；Vesicle film is can The biodegradable of inverse crosslinking and the PTMC of good biocompatibility, it is pungent that the dithiolane of side chain is similar to human body Natural antioxidant sulfur Acid, it is possible to provide the reversible crosslink that reduction is sensitive, not only supports Nano medication long circulating in blood, it is also ensured that intracellular soon Speed solves crosslinking, in release nucleic acid to target cell；Shell has targeted molecular with PEG for background simultaneously, can be high to cancerous cell Specific binding；The nano-scale of vesicle and tumour-specific targeting make vesicle can carry nucleic acid, and efficiently to enter tumor thin Born of the same parents.

2. the present invention can be for loading complex function by having the cross linked polymer vesicle of the spermine of positively charged to inside Property siRNA and DNA medicine, its inside and outside have significant Gene silencing efficacy.Spermine used herein is that organism is natural to be deposited Polyamines, nontoxic, imitated vesicle structure can be formed after combining PEG chain segment and hydrophobic segment, there is good drug encapsulation effect； The polymer support of the present invention avoids existing non-viral cationic polymer by electrostatic interaction bind nucleic acid shape simultaneously Instability that the complex become brings, positively charged easily with Cell binding and migration force is poor, release efficiency is poor defect.

3. the inner membrance of the present invention have positive charge, the sensitive reversible crosslink of reduction, the intracellular biology solving crosslinking can drop Depolymerization compound vesicle carrier avoids that existing protein nano carrier load efficiency is low, protein changeableness or protein are released The defect that bioavailability is low, drug effect is low caused such as slow down；The polymer support of the present invention avoids existing at physiology simultaneously Small-molecule drug electronegative under environment such as pemetrexed, methotrexate etc. lack suitable carrier or efficiency of loading is low, release Slow down, the defect such as the bioavailability of medicine is low, drug effect is low.

4. this vesicle system has the advantage of multiple uniqueness, including the most handling, the outstanding bio-compatible of preparation Property, to medicine fabulous control release property, superpower body-internal-circulation stability, to the superior targeting of cancerous cell, significantly special Specific gene silence energy, the ability of remarkable suppression growth and metastasis of tumours.Therefore, the vesicle of the present invention is expected to become defecate collection The Nano medication platform such as prompt, stable, multi-functional, for efficiently, active targeting conveying protein, nucleic acid and physiologic ring The electronegative small-molecule drug in border is to tumor.

Accompanying drawing explanation

Fig. 1 is PEG5k-P (DTC4.6k-TMC13.5k)-SP nuclear magnetic spectrogram in embodiment one；

Fig. 2 is DP in embodiment three₈-PEG6.5k-P (DTC6k-TMC15k) nuclear magnetic spectrogram；

Fig. 3 is the targeting crosslinking stability of vesicle, TEM, reduction response diagram in embodiment eight；

Fig. 4 is to carry FITC-CC in embodiment 11 to cross-link vesicle FITC-CC-DP₈The release figure of/RCCPs；

Fig. 5 is that embodiment 17 hollow cross-links vesicle DP₈/ the RCCPs toxicity figure to MCF-7 breast carcinoma cancerous cell；

Fig. 6 is to carry granzyme GrB in embodiment 18 to cross-link vesicle GrB-DP₈/ RCCPs is to MCF-7 breast cancer cell, HepG2 The cytotoxicity figure of hepatoma carcinoma cell；

Fig. 7 is to carry FITC-CC in embodiment 19 to cross-link vesicle FITC-CC-DP₈/ RCCPs to MCF-7 breast cancer cell, The cell CLSM figure of HepG2 hepatocarcinoma；

Fig. 8 is to carry CC-Cy5 in embodiment 20 to cross-link vesicle CC-Cy5-DP₈/ RCCPs efficacy of a drug kinetics in Mice Body is bent Line chart；

Fig. 9 is to carry CC-Cy5 in embodiment 21 to cross-link vesicle CC-Cy5-DP₈/ RCCPs is to MCF-7 breast carcinoma subcutaneous tumor mould The bio distribution figure of type；

Figure 10 is to carry CC-Cy5 in embodiment 22 to cross-link vesicle CC-Cy5-DP₈/ RCCPs is to MCF-7 breast carcinoma subcutaneous tumor mould The image of type；

Figure 11 is to carry pemetrexed crosslinking vesicle PEM-CC9-RCCPs lung cancer therapy subcutaneous to lotus H460 in embodiment 23 in fact Testing figure, wherein respectively organize tumor growth curve during A treatment, B is group tumor figure each after treatment, and C is body weight change curve.

Detailed description of the invention

Below in conjunction with embodiment and accompanying drawing, the invention will be further described:

Embodiment one synthetic polymer PEG5k-P (DTC4.6k-TMC13.5k)-SP

Synthesis is divided into two steps, is first that ring-opening polymerisation prepares PEG5k-P (DTC4.6k-TMC13.5k) diblock copolymer, specifically Operate as follows, in a nitrogen environment, weigh successively MeO-PEG-OH (M _n =5.0 kg/mol, 0.20 g, 40 μmol), TMC (0.6 g, 5.9 mmol) and DTC (0.192g, 1.0 mmol) is also dissolved in dichloromethane (DCM, 6.8 mL) In, rapidly join ring-opening polymerization catalyst, such as double (double trimethyls are silica-based) amine zinc (7.7 mg, 20 μm ol).Closed reactor Good seal is placed in 40 ° of C oil baths and is reacted 24 hours under magnetic agitation.Glacial acetic acid terminate reaction after in ice ether precipitate twice, Sucking filtration, normal-temperature vacuum obtain product after drying.Productivity: 90.3%.¹H NMR (400 MHz, DTCl₃): PEG: δ 3.38, 3.65; TMC: δ 4.24, 2.05;DTC: δ 4.32,3.02, the molecular weight understanding each section of polymer is calculated through nuclear-magnetism For 5.0-(4.6-13.5) kg/mol.GPC records molecular weight distribution: 1.39.

Then, terminal hydroxyl and the p-nitrophenyl chloroformate ester NPC of the PEG5k-P (DTC4.6k-TMC13.5k) obtained are anti- Should activate, then with spermine (M _n=202.34 kg/mol) primary amine reaction prepare.Concrete, PEG5k-P (DTC4.6k- TMC13.5k) (0.2g, hydroxyl 0.0087 mmol) and pyridine (3.5 L) are dissolved in dry dichloromethane (DCM), ice bath Under be slowly added dropwise the DCM solution of NPC (9.2mg, 0.046 mmol), rear room temperature reaction 12 hours, then precipitate in ice ether Twice, filter, be vacuum dried and obtain PEG5k-P (DTC4.6k-TMC13.5k)-NPC.It follows that product to be dissolved in 3 mL DCM After be added drop-wise in the 2mL DCM dissolved with spermine (SP, 34.7 mg, 0.172 mmol), react at 25 DEG C after 24 hours at DCM and Dialysis (MWCO 3500) 48 hours in methanol (volume ratio is 1:1), precipitate twice, sucking filtration room temperature true in concentration, ice ether Sky is dried to obtain product PEG5k-P (DTC4.6k-TMC13.5k)-SP.Productivity: 87.3%.Accompanying drawing 1 is PEG5k-P (DTC4.6k- TMC13.5k) nuclear magnetic spectrum of-SP, its¹H NMR (400 MHz, DTCl₃) characterize display except PEG and P (DTC-TMC) peak (PEG: δ 3.38,3.65 outward; TMC: δ 4.24, 2.05;, the characteristic peak of also spermine DTC: δ 4.32,3.02) At 2.6-2.8 and 3.23.

Embodiment two synthetic segmented copolymer Mal-PEG6k-P (DTC4.8k-TMC15.2k)-SP

The synthesis of Mal-PEG6k-P (DTC4.8k-TMC15.2k)-SP is similar with embodiment one, is also divided into two steps, simply will Initiator MeO-PEG-OH in the first step therein is changed to maleimide-functionalised Mal-PEG6k-OH, ring-opening polymerisation TMC and DTC obtains Mal-PEG6k-P (DTC4.8k-TMC15.2k), and then its terminal hydroxyl NPC activates, then with spermine Primary amine reaction prepares.Concrete operations are similar with embodiment one.Productivity: 90.2%.1H NMR (400 MHz, DTCl3): PEG: δ 3.38, 3.65; TMC: δ 4.24, 2.05;DTC: δ 4.32,3.02, and Mal and the characteristic peak of spermine.Polymer Number-average molecular weight is 6.0-(4.8-15.2)-0.2 kg/mol by characteristic peak area integration ratio calculation.

Embodiment three synthesizes targeting diblock polymer DP8-PEG6.5k-P (DTC6k-TMC15k)

The synthesis of DP8-PEG6.5k-P (DTC6k-TMC15k) is similar with embodiment one, is also divided into two steps.The first step will be real Execute the initiator MeO-PEG-OH in the first step of example one and be changed to the NHS-PEG6.5k-OH of N-hydroxy-succinamide functionalization, Ring-opening polymerisation TMC and DTC obtains NHS-PEG6.5k-P (DTC6k-TMC15k), i.e. under nitrogen environment, order in closed reactor Add 0.15 g (0.781 mmol) DTC, the TMC of 0.43 g, 0.2 g (0.0154 mmol) NHS-PEG6.5k- OH and the DCM of 5 ml, dissolves, and is subsequently adding the ring-opening polymerization catalyst such as double (double three of 5.9 mg (0.0154 mmol) Methylsilyl) amine zinc, post processing with embodiment one.Nuclear-magnetism calculates NHS-PEG6.5k-P (DTC6k-by integral area TMC15k).Second step, polypeptide DMAPTVLP (DP8) is according to its amino and NHS-PEG6.5k-P (DTC6k-TMC15k) mol ratio 3:1 adds, 30oC amidation process 24-72 hour, and dialysis removes free DP8, and lyophilization obtains DP8-PEG6.5k-b-P (DTC6k-TMC 15k), calculates DP8 percent grafting close to 100% by nuclear-magnetism (accompanying drawing 2) and TNBSA.

Embodiment four synthesizes targeting diblock polymer cNGQ-PEG7k-P (DTC4.8k-TMC19.2k)

The synthesis of cNGQ-PEG7k-P (DTC2.8k-TMC14.2k) is similar with embodiment one, is also divided into two steps, by the first step In initiator MeO-PEG-OH be changed to the NHS-PEG-OH, ring-opening polymerisation TMC and DTC of N-hydroxy-succinamide functionalization and obtain To NHS-PEG7k-P (DTC4.8k-TMC19.2k)；Secondly, there is peptide C (NGQGEQ) (cNGQ) and the NHS-of free primary amine PEG7k-P (DTC4.8k-TMC19.2k) is bonded by amidation process.Briefly, NHS-PEG7k-P (DTC4.8k- TMC19.2k) (0.5 g, 0.017 mmol) and cNGQ (20 mg, 0.033 mmol) are dissolved in 5 mL DMF in succession In, normal-temperature reaction is after 2 days, and dialyse in distilled water two days (MWCO 3500), and lyophilization obtains product cNGQ-PEG7k-P (DTC4.8k-TMC19.2k).Productivity: 81.2%.1H NMR (400 MHz, DMSO-d6): PEG: δ 3.51; TMC: δ 4.23, 1.94; DTC: δ 4.13, 2.99; cNGQ: δ 6.84–7.61.BCA protein reagent box (Thermo Scientific) percent grafting recording cNGQ is 89.7%.By use, there are the available different targeting of different active PEG to divide The polymer of son.

Embodiment five synthesizes polymer A zide-PEG6.5k-P (DTC4.0k-LA15.3)-SP

The synthesis of Azide-PEG6.5k-P (DTC4.0k-LA15.3)-SP is similar with embodiment one, is also divided into two steps, simply The initiator MeO-PEG-OH of its first step is changed to azide-functionalized Azide-PEG6.5k-OH, ring-opening polymerisation LA and DTC Obtaining Azide-PEG6.5k-P (DTC4.0k-LA15.3), then its terminal hydroxyl NPC activates, more anti-with the primary amine of spermine Should prepare.Concrete operations are similar with embodiment one.Productivity: 90.2%.¹H NMR (400 MHz, DTCl₃): PEG: δ 3.38, 3.65; TMC: δ 4.24, 2.05;DTC: δ 4.32,3.02, and the characteristic peak of spermine.Polymer number-average molecular weight It is 6.5-(4.0-15.3)-0.2 kg/mol by characteristic peak area integration ratio calculation.

The material rate used by adjustment can get the polymer of different molecular weight, is shown in Table 1.

Each polymer preparation condition of table 1 and the nuclear-magnetism characterization result of product

Embodiment six prepares cross linked polymer vesicle PEG5k-P (DTC4.6k-TMC13.5k)-SP

Prepared by solvent displacement.To the HEPES(5 mM, pH 6.8 of 950 μ L under room temperature) to add 50 μ L concentration in buffer be 5 The DMSO solution of PEG5k-P (the DTC4.6k-TMC13.5k)-SP of mg/ml, room temperature stands 1h, slowly rotates mixed liquor, After being allowed to be uniformly dispersed, (no) adds and is equivalent to after the DTT solution (final 0.1 mM) of DTC mole 10%-30% 37^oC shakes 12h fully (certainly) crosslinking is shaken in Chuan.Then dialysis (MWCO:3500) 12h removes organic solvent and floating preteins, and period changes 5 media, thus obtaining can reciprocal kernel crosslinking vesicle, referred to as RCCPs.

The synthesis of embodiment seven target polymer and the preparation of target polymer vesicle

The synthesis of target polymer has various ways.

The Alkynyl-PEG5k-OH of alkynes functionalization causes DTC Yu LA ring-opening polymerisation, terminal hydroxy group activation and spermine to react To Alkynyl-PEG5k-P (the DTC5.8k-LA23k)-SP that end is activity alkynyl；Finally, the targeting with azide functionalization divides Son, such as polypeptide cNGQ-N3 or galactose Gal-N3, is reacted by the click chemistry of nitrine-alkynyl and obtains target polymer Gal- PEG5k-P(DTC5.8k-LA23k)-SP.Then the preparation of targeting vesicle be i.e. Gal-PEG5k-P (DTC5.8k-LA23k)- SP and PEG5k-P (DTC5.8k-LA23k)-SP squeezes in HEPES solution after being blended in DMSO, prepares with embodiment six Gal-RCCPs。

The Azide-PEG3k-OH of azide functionalization causes DTC Yu TMC ring-opening polymerisation, terminal hydroxy group activation to react with spermine Obtain Azide-PEG3k-P (the DTC4k-TMC12k)-SP that end is reactive azido, finally, with the targeting of alkynyl functionalization Molecule, such as alk-CC9 or cRGD-alk, obtains target polymer CC9-PEG3k-P by the click chemistry of nitrine-alkynyl (DTC4k-TMC12k)-SP.Then the preparation of targeting vesicle be i.e. CC9-PEG3k-P (DTC4k-TMC12k)-SP and PEG3k-P (DTC4k-TMC12k)-SP squeezes in HEPES solution after being blended in DMSO, prepares CC9-with embodiment six RCCPs。

The polymer of nitrine or alkynyl functionalization is the diblock polymer without end spermine, i.e. Alkynyl-PEG5k-P (DTC5.8k-LA23k) and Azide-PEG3k-P (DTC4k-TMC12k), the mode of the polypeptide such as its bonding cNGQ and prepare targeting The mode of vesicle (mixing with the triblock polymer without targeting) is similar with above-mentioned example.

The Mal-PEG6k-OH or acrylic ester functionalized AA-PEG6.5k-OH of maleimide Mal functionalization draw Send out DTC Yu TMC ring-opening polymerisation, terminal hydroxy group activation is reacted with spermine and is obtained polymer Mal-PEG6k-P (DTC4.8k- TMC19.2k)-SP or AA-PEG6.5k-P (DTC4.6k-TMC18.6k)-SP.Then, they and corresponding inactive end is poly- After compound PEG5k-P (DTC4.6k-TMC18.6k)-SP mixing is dissolved in DMSO, squeezes in HEPES solution, make with embodiment six Standby obtaining cross-links vesicle.Finally, vesicle solution adds targeted molecular such as polypeptide cNGQ-SH or folic acid containing free sulfhydryl group FA-SH or CPP33-SH, is bonded by the vesicle of active Mal or AA of Michael addition reaction and surface, obtains targeting polymerization Thing vesicle CPP33-RCCPs, FA-RCCP etc..

Maleimide Mal and acrylic ester functionalized block polymer are the diblock polymer without end spermine, I.e. Mal-PEG6k-P (DTC3.2k-TMC15.4k) and AA-PEG5k-P (DTC4.5k-TMC19.3k), itself and end have spermine Triblock polymer be mixed with vesicle, the mode of the bonding polypeptide such as cNGQ and prepare the mode of targeting vesicle and above-mentioned example Similar.

Embodiment eight prepares targeting self-crosslinking polymer vesicle DP8-RCCPs

Prepared by solvent displacement.Target polymer vesicle is prepared by several ways.The first is based on triblock polymer and target To diblock polymer.Such as, the DP of embodiment three preparation₈-PEG6.5k-P (DTC6k-TMC15k) and PEG5k-P (DTC4.6k-TMC13.5k)-SP mixes in specific proportions, is dissolved in DMSO, joins HEPES(5 mM, pH 6.8) buffer In, stand with embodiment six, cross-link, dialyse, obtain targeting crosslinking vesicle, be labeled as DP8-RCCPs.Target polymer DP₈- The content of PEG6.5k-b-P (DTC6k-TMC15k) is 5 ~ 40wt.%.DLS measures the self-crosslinking polymer vesicle size of preparation About 80-125nm, particle diameter is distributed as 0.11-0.16.Fig. 3 is the distribution of above-mentioned self-crosslinking vesicle particle diameter and the test (A) of stability And transmission electron microscope(TEM) picture (B), crosslinking vesicle and reduction response test (C)；The size of the self-crosslinking vesicle obtained by The nanoparticle vesicle of the formation that dynamic light scattering particle size analyser (DLS) is surveyed is 80 ~ 125nm, and particle diameter distribution is the narrowest, and high power is dilute Constant particle diameter and particle diameter distribution (Fig. 3 A) is remained in that after releasing；The nanoparticle recorded from Fig. 3 B, TEM is hollow Imitated vesicle structure；But quickly discharge under simulation tumor cell reducing environment, solve crosslinking (Fig. 3 C).It follows that the vesicle obtained Self-crosslinkable, and there is the character solving crosslinking that reduction is sensitive.

Embodiment nine prepares targeting self-crosslinking polymer vesicle cRGD-RCCPs

Target polymer vesicle is prepared based on two kinds of triblock polymers.Such as, the two kinds of PEG5k-P mixed in specific proportions (DTC4.6k-TMC13.5k)-SP and cRGD-PEG6.5k-P (DTC6k-TMC15k)-SP is dissolved in DMSO, joins HEPES In (5 mM, pH 6.8) buffer, stand with embodiment six, add that to be equivalent to the DTT solution of DTC mole 10%-30% (final 0.1 mM), dialysis, obtain targeting crosslinking vesicle, be labeled as cRGD-RCCPs.Depend on the ratio of different target polymer and gather The dissolution time of compound, the vesicle mean diameter obtained is about 50-125nm, and particle diameter is distributed as 0.04-0.17.There is vesicle Feature, stabilization in vitro, there is reduction-sensitive.

Embodiment ten prepares targeting self-crosslinking polymer vesicle FA-RCCPs

Based on triblock polymer and the two of active group or triblock polymer, such as, the Mal-PEG6k-P of embodiment two (DTC4.8k-TMC15.2k)-SP and PEG5k-P (DTC4.6k-TMC13.5k)-SP mixes in specific proportions and is dissolved in DMSO, adds Enter to HEPES(5 mM, pH 6.8) in buffer, stand with embodiment six, cross-link, dialyse, obtain cross-linking vesicle.Then, logical Cross Michael's addition and after room temperature reaction, obtain targeting crosslinking vesicle with the targeted molecular such as folic acid FA-SH containing free sulfhydryl group, It is labeled as FA-RCCPs.Depend on ratio and the dissolution time of polymer, the vesicle average particle obtained of different target polymer Footpath is 58-130nm, and particle diameter is distributed as 0.06-0.17.Vesicle stabilization in vitro, there is reduction-sensitive.

Embodiment 11 preparation loads protein or the crosslinking vesicle of DNA and targeting crosslinking vesicle

Carry protein such as cytochrome C, granzyme B and DNA cross-link vesicle preparation with embodiment seven, simply HEPES solution In be dissolved with protein or the DNA of variable concentrations in advance.Such as carry the cell color of the FITC labelling of different proportion (2-30wt.%) Element C(FITC-CC) the particle diameter of cross linked polymer vesicle at 90-108 nm, particle diameter is distributed in the vesicle that 0.13-0.19. obtains For FITC-CC-DP8-RCCPs.The parcel efficiency of its fluorescent spectrophotometer assay FITC-CC is 50%-100%.

Granzyme B (GrB) is identical with the loading of DNA.Medicine carrying targeting vesicle is prepared with embodiment eight, nine, ten, simply HEPES solution is dissolved with protein or the DNA of variable concentrations in advance, obtains GrB-DP8-RCCPs and DNA-DP8-RCCPs.

Release in vitro uses dialysis.Illustrate as a example by the release in vitro of FITC-CC, 37^oC constant-temperature table shakes (200rpm) carrying out, often group is respectively arranged with three Duplicate Samples.First group, the cross linked polymer vesicle carrying FITC-CC adds 10mM Reducing environment PB(10Mm of DTT, pH 7.4) in；Second group, carry FITC-CC polymer vesicle PB (10Mm, Ph7.4) in；The concentration of medicine carrying crosslinking vesicle is 50mg/L, takes 0.5 ml and puts in bag filter in (MWCO:350kDa), often Individual test tube adds corresponding dialysis medium 25ml, takes the outer medium of 5.0ml bag filter in the scheduled time and be used as test, add 5.0ml fresh medium.Fluorescence spectrophotometer measures drug concentration in solution.Accompanying drawing 4 is cumulative release amount and the time of FITC-CC Relation, it can be seen that in addition analog cell after DTT, its release is signifi-cantly more rapidly than the sample not adding DTT, illustrates that medicine carrying cross-links Vesicle, in the presence of the DTT of 10 mM, can effectively discharge medicine.

Embodiment 12 preparation loads crosslinking vesicle and the targeting crosslinking vesicle of DNA

The DNA-RCCPs of parcel DNA can be prepared by solvent exchange method.DNA is pcDEF3-CD8IL-36 γ (pIL-36 γ), Or calf thymus DNA etc. pcDEF3-CD8IL-12(pIL-12).Such as PEG5k-P (DTC3.0k-TMC15k)-SP prepares DNA- Under RCCPs concrete operations are, the DMSO solution (5.0 mg/mL) of 100 μ L PEG5k-P (DTC3.0k-TMC15k)-SP is slow Squeeze into 900 μ L dissolved predetermined quantity DNA (1 mg/mL) HEPES (10 mM, pH 6.8) mixed solution in, room Temperature stands overnight, dialyse, add catalytic amount reducing agent dithiothreitol (DTT) crosslinking obtain DNA-RCCPs.DLS result shows, DNA-RCCPs particle diameter increases along with the increase of DNA content, is shown in Table 2.In vivo test content is the sample of 20wt%DNA later Product.

The particle diameter of table 2 DNA-cNGQ/RCCPs and the relation of DNA content

Embodiment 13 loads the gel electrophoresis analysis of the crosslinking targeting vesicle DNA-cNGQ-RCCPs of DNA

DNA-cNGQ/RCCPs is prepared with embodiment 12.According to weight ratio 4:1 mixing PEG5k-P (DTC4.4k- TMC19.8k)-SP and cNGQ-PEG7k-P (DTC4.8k-TMC19.2k).DNA have employed pcDEF3-CD8IL-36 γ (pIL- 36 γ) and pcDEF3-CD8IL-12(pIL-12).0.7 g agarose is added in 70 mL tetrabromoethane (TBE) buffer solution, Heating for dissolving agarose powder, adds 1 μ L ethidium bromide after the cooling period, obtains agarose gel stand-by.DNA-cNGQ-RCCPs or DNA-RCCPs DNA and polymer percentage by weight (wt .%) it is respectively set as 10%, 20%, 30%, 40%, 50%.At glue In be separately added into the DNA-cNGQ-RCCPs of 20 μ L, DNA-RCCPs, free DNA, in TBE electrophoretic buffer run glue (100 V, 30 min).Afterwards, Molecular Imager FX (Bio-Rad, Hercules, Ex/Em:532/605 nm) clap According to gel images, analyzed by Quantity One software (Bio-Rad).Agarose gel detention test result indicate that, even if When DNA content reaches 50 wt.%, cNGQ-RCCPs still can wrap up DNA by consolidation completely, it was demonstrated that DNA-cNGQ-RCCPs stability is excellent Different.Result display vesicle can effectively be combined DNA(pIL-12).

Embodiment 14 preparation carries crosslinking vesicle and the targeting crosslinking vesicle siRNA-RCCPs of siRNA

The crosslinking vesicle loading siRNA is prepared by solvent exchange method.Such as, load without specific comparison siRNA (siScramble) method obtaining siRNA-RCCPs.The HEPES buffer solution of predetermined quantity siRNA (1 mg/mL, 10 MM, pH 7.4) and DMSO solution (5.0 mg/mL) mixing of 100 μ L PEG5k-P (DTC4.4k-TMC19.8k)-SP, then Squeeze into the HEPES (10 mM, pH 6.8) of 900 μ L, left at room temperature over night, HEPES dialyse, hatch 4 h crosslinking obtain siRNA-RCCPs.DLS result display parcel 10wtDuring % siRNA, particle diameter is 100 ran, and TEM confirms wherein Hollow structure.Table 3 is the particle diameter relation with siRNA content of siRNA-cNGQ/RCCPs；Along with siRNA content increases to 50 from 0wt .The particle diameter of %, siRNA-cNGQ/RCCPs is also risen to 175 nm by 109.Later cell experiment and in vivo test such as without It is the sample of 10wt% that specified otherwise is all tested at siRNA.

The particle diameter of table 3 siRNA-cNGQ/RCCPs and the relation of siScramble content

The vesicle loading fluorescent labeling Cy5-siRNA or luciferase gene labelling siGL3 or therapeutic siPLK1 is similar. Load the most commensurability siRNA(10%-80%) time, particle diameter is 90-180 nanometer.

The gel electrophoresis analysis of embodiment 15 siScramble-cNGQ/RCCPs

As embodiment 14 prepares siScramble-cNGQ/RCCPs, as embodiment 13 prepares gel and runs glue, siRNA- CNGQ/RCCPs or siRNA-RCCPs siRNA and polymer percentage by weight (wt .%) be respectively set as 10%, 20%, 30%, 40%, 50%, 60%, 70% and 80%.Glue is separately added into the siRNA-cNGQ/RCCPs of 20 μ L, siRNA-RCCPs, from By siRNA, and process the siRNA-cNGQ/RCCPs after 20 h with 10 mM GSH.Test result indicate that, even if working as siRNA Content is up to 80wt .%, cNGQ/RCCPs still can completely, consolidation parcel siRNA, it was demonstrated that siRNA-cNGQ/RCCPs is stable Property excellent.But, after it hatches 20 h in the presence of 10 mM GSH find, due to crosslinking vesicle solution crosslinking and swelling greatly Part siRNA discharges.

Embodiment 16 preparation carries crosslinking vesicle and the targeting crosslinking vesicle PEM-RCCPs of pemetrexed

The crosslinking vesicle loading physiological environment electronegative small-molecule drug such as pemetrexed is prepared by solvent exchange method, with reality The bag executing protein in example 11 carries similar.By polymer P EG5k-P (the DTC4.4k-TMC19.8k)-SP's of 100 μ L DMSO solution (5.0 mg/mL) squeeze into the pemetrexed containing predetermined quantity of 900 μ L HEPES buffer solution (1 mg/mL, 10 mM, pH 6.6), left at room temperature over night, HEPES dialyse, hatch 4 h crosslinking obtain PEM-RCCPs.Carry 20 wt% trains The particle diameter of the cross linked polymer vesicle of beautiful Qu Sai at 90 nm, particle diameter be distributed in the vesicle that 0.14. obtains for PEM-RCCPs. It is 89% that its ultraviolet spectrophotometer measures the parcel efficiency of PEM.

Embodiment 17 mtt assay surveys the vesicle toxicity to breast cancer cell MCF-7

Mtt assay uses human breast carcinoma cancerous cell (MCF-7), with 5 × 10³Individual/mL by cell kind in 96 orifice plates, every hole 80 μ L, 24 Cultivate after hour to cell attachment about 70%.The preparation of cross linked polymer vesicle is prepared by embodiment seven, eight.Then, experimental group Each hole is separately added into the vesicle containing variable concentrations (0.1-0.5 mg/mL), separately sets cell blank control wells and culture medium is empty White hole (multiple 4 holes).After cultivating 24 hours, every hole adds MTT(5.0 mg/mL) 10 μ L, after continuing to cultivate 4 hours, every hole adds 150 μ L DMSO dissolve crystallization generated, and survey absorbance (A) by microplate reader, adjust with culture medium blank well at 492 nm Zero, calculate cell survival rate.In Fig. 5, result shows, when the concentration of cross linked polymer vesicle increases to 0.5 mg/mL from 0.1, The survival rate of MCF-7 remains above 85%, illustrates that this cross linked polymer vesicle has good biocompatibility.

Additionally being prepared for surface and have the crosslinking vesicle of different targeted molecular, such as, ibid mtt assay have studied FA system pair Ovarian cancer SKOV-3 cell, the effect of human mouth epidermoid carcinoma cell KB cell, cNGQ system is to lung cancer A549 cell, Gal body System is to hepatocellular carcinoma H22, and the cytotoxicity that cRGD system is to Melanoma B16 cell, and result also illustrate that these empty friendships Connection targeting vesicle has good biocompatibility.

Embodiment 18 mtt assay surveys the targeting crosslinking vesicle toxicity to breast cancer cell MCF-7 carrying GrB

Test object is embodiment 11 GrB-DP8-RCCPs, with without targeting and 5%, the medicine carrying of 10%, 20%, 30%, 40% targeting The vesicle research toxicity to MCF-7 cell, GrB concentration range is 0.001,0.01,0.05,0.1,0.2,0.32 μ g/ mL.The cultivation of cell is with embodiment 17, and co-cultivation is after 4 hours, and sucking-off sample replaced with fresh medium continues to hatch 68 h After, MTT then adds, processes and measure absorbance with embodiment 17.We have also done drug-carrying polymer vesicle to polypeptide The toxicity test of the MCF-7 breast cancer cell of end-blocking, is initially charged free polypeptide DP8 and hatches 4h before adding drug-carrying nanometer particle, more same Above-mentioned experimental implementation is identical.From result in Fig. 6 A, B, carry GrB containing 30%DP₈Targeting cross linked polymer vesicle is to MCF-7 Half lethal concentration (the IC of cell₅₀) it is 0.188 μ g/mL, far below the half lethal concentration without targeting vesicle, illustrate the present invention's Medicine can be well sent to intracellular by vesicle, and effectively discharges, and finally kills cancerous cell, and the effect of targeted nano granule Want more preferably, and the cell survival rate blocked is all more than 70%.It addition, cell surface paranuclein is expressed low by medicine carrying crosslinking vesicle The toxicity of HepG2 hepatoma carcinoma cell poor, result in Fig. 6 C show, paranuclein MCF-7 cell surface special expression with And the good targeting of DP8.

Be prepared for surface have different targeted molecular crosslinking vesicle, load pemetrexed, methotrexate, cytochrome C, Or apoptosis element etc., by mtt assay research FA system to ovarian cancer SKOV-3 cell, the work of human mouth epidermoid carcinoma cell KB cell With, cNGQ system is to lung cancer A549 cell, and Gal system is to hepatocellular carcinoma H22, and cRGD system is to Melanoma B16 cell Cytotoxicity, all embody the specificity of targeting, the target polymer vesicle of medicine carrying can enter cell faster and play effect Really.

The endocytosis of embodiment 19 target drug-carrying vesicle and intracellular release experiment

The endocytosis of target drug-carrying vesicle and intracellular release experiment as a example by medicine carrying vesicle FITC-CC-DP8-RCCPs, use swash Light Laser Scanning Confocal Microscope (CLSM) tracking and measuring.By the DMEM of the MCF-7 cell of 450 L and HepG2 cell (containing 10% Ox blood serum, 100IU/ml penicillin and 100 g/ml streptomycins) suspension is laid on 24 well culture plates (every hole 5 × 10⁴Individual cell) in, 37 DEG C, 24h is cultivated under 5% carbon dioxide conditions.The PBS solution of FITC-CC-RCCPs and FITC-CC-DP8-RCCPs of 50 L is added In hole (final concentration of 10 g/ml of FITC), after then continuing to hatch 4h, 8h, 12h, remove culture medium and wash three times with PBS, use Paraformaldehyde solution 200 L of 4% fixes 15min, PBS and washes 3 times.Finally use CLSM(TCS SP5) observe and take pictures.By Fig. 7 result Show that FITC-CC-DP8-RCCPs (Fig. 7 A8h, B12h) is relative to without targeting FITC-CC-RCCPs(Fig. 7 C 12h) can lead to Cross mediation more effective endocytosis entrance MCF-7 cell and FITC-CC can quickly discharge intracellular, cause effective cell to wither Die, and HepG2 cell be there is no Targeting Effect (Fig. 7 D).

It is prepared for surface to there is the medicine carrying crosslinking vesicle of different targeted molecular, utilize CLSM experimentation FA system to ovary Cancer SKOV-3 cell, the effect of human mouth epidermoid carcinoma cell KB cell, cNGQ system is to lung cancer A549 cell, Gal system pair Hepatocellular carcinoma H22, and cRGD system is to the cell endocytic of Melanoma B16 cell and intracellular release behavior, the equal table of result Bright target polymer thing vesicle can faster, more effectively by target cell annex rapid delivery of pharmaceuticals.

The blood circulation of embodiment 20 Cy5-CC-RCCPs and Cy5-CC-DP8-RCCPs crosslinking vesicle

All zooperies operation meets University Of Suzhou's animal experimental center regulation and strictly carries out.Experiment selects body weight to be 18 ~ 20 About gram, the Balb/C nude mice of 4 ~ 6 week old.First prepared the albumen of Cy5 labelling by amidation process with Cy5-NHS and CC Cy5-CC.Vesicle Cy5-CC-DP8-RCCPs(130 nanometer, particle diameter is distributed as 0.17) and quiet without targeting Cy5-CC-RCCPs tail In arteries and veins injection Mice Body (Cy5 concentration is 4 μMs), take blood about 10 μ 0,0.25,0.5,1,2,4,8,12 and 24 hours fixed points L, accurately calculates blood weight by difference assay, then add 100 μ L, concentration be 1% TritonX and the extraction of 500 μ L dimethyl sulfoxides (wherein containing the DTT of 20 mM)；It is then centrifuged for (20000 revs/min, 20 minutes) and takes the supernatant, surveyed by fluorescence spectrophotometer Obtain the amount of each time point Cy5.From calculating, targeting crosslinking vesicle, non-targeted crosslinking vesicle elimination in Mice Body half The phase of declining is respectively 4.36 and 3.33 hours, so the crosslinking vesicle of the present invention is stable in Mice Body, has longer cycle times, as Shown in Fig. 8.

Have studied several representative sample circulation time in Mice Body, result shows, the PEG8k-P of medicine carrying (DTC8k-LA30k)-SP crosslinking vesicle, PEG7k-P (DTC4k-LA18k)-SP crosslinking vesicle and PEG3k-P (DTC0.9k- TMC6k)-SP crosslinking vesicle Chinese medicine blood circulation time in Mice Body is respectively 7.56 hours, 6.51 hours and 2.18 Hour.

Embodiment 21 carries protein-crosslinking vesicle Cy5-CC-RCCPs and Cy5-CC-DP8-RCCPs at lotus MCF-7 breast The vivo biodistribution distribution of adenocarcinoma mice

Animal is with embodiment 20.At subcutaneous injection 1 × 10⁷Individual MCF-7 cell, after about 3 ~ 4 weeks, tumor size is 100 ~ 200 mm³Time start experiment.By Cy5-CC-DP8-RCCPs, free PROTEIN C y5-CC and non-targeted Cy5-CC-RCCPs tail vein In injection Mice Body (Cy5-CC:0.25 mg equiv./kg), after 8 hours, put to death mouse, by tumor and the heart, liver, spleen, lung and Nephridial tissue is taken out, and cleaning is added the TritonX of 500 μ L 1% and ground by refiner after weighing, add 900 μ L diformazans sub- Sulfone extraction (the wherein DTT containing 20 mM).Centrifugal (20000 revs/min, 20 minutes) take supernatant, when fluorescence spectrophotometer is surveyed each Between put the amount of Cy5-CC.As shown in Figure 9, Cy5-CC-DP8-RCCPs and Cy5-CC-RCCPs injects 8 hours in tumor accumulation Cy5-CC amount is respectively 9.5 and 5.4 ID%/g, and the former is 1.76 times of the latter, illustrates that Cy5-CC-DP8-RCCPs is by actively Targeting is more in tumor accumulation.

Embodiment 22 medicine carrying crosslinking vesicle bubble Cy5-CC-RCCPs and Cy5-CC-DP8-RCCPs is at MCF-7 mammary gland The living imaging experiment of cancer mice

Animal selects with embodiment 20.In Cy5-CC-RCCPs and Cy5-CC-DP8-RCCPs Tail Vein injection Mouse body, Time point follows the trail of the whereabouts of vesicle for 4,8,12,24 hours with small animal living body imager.Experimental result as shown in Figure 10, Cy5- CC-DP8-RCCPs quickly accumulates at tumor locus, and fluorescence is the strongest after 24 hours.Cy5-CC-DP8-RCCPs is described Can active targeting and be enriched to breast cancer tumour position, breast cancer cell is had extremely strong specificity.And medicine carrying Cy5-CC- RCCPs crosslinking vesicle quickly metabolism after entering tumor, fluorescence intensity is low.

Embodiment 23 medicine carrying targeting crosslinking vesicle PEM-CC9-RCCPs is in the mice of the subcutaneous pulmonary carcinoma of lotus H460 Tumor killing effect, body weight change and survival rate

Animal selects with embodiment 20, at subcutaneous injection 1 × 10⁷Individual H460 human lung carcinoma cell, after about 3 ~ 4 weeks, tumor is big Little is 100 ~ 200 mm³Time start experiment.PEM-CC9-RCCPs, PEM-RCCPs, clinical injection liquid Alimta and PBS 0, 4, within 8 and 12 days, (PEM:12.5 mg/kg in Tail Vein injection Mouse body is passed through；Alimta:25 mg/kg).At 0 ~ 18 day, every two It weighs the body weight of mice, vernier caliper measurement gross tumor volume, and gross tumor volume computational methods are: V=(L × W × H)/2, (wherein L For the length of tumor, W is the width of tumor, and H is the thickness of tumor).The existence of continuous observation mice to 60 days.By can in Figure 11 Know, PEM-CC₉During-RCCPs treatment group 20 days, tumor is significantly suppressed, and medicine carrying PEM-RCCPs group tumor has certain increasing Long.By contrast, the Mouse Weight of PEM-CPP-RCCPs and PEM-RCCPs group, almost without change, illustrates that medicine carrying cross-links vesicle Mice do not had toxic and side effects.PEM-CC₉-RCCPs treatment group is the most all survived, and Alimta group is whole when 38 days Death, PBS group is also the most dead when 30 days.Therefore, tumor can effectively be suppressed after the targeting crosslinking vesicle medicine carrying of the present invention Increase, mice is not had toxic and side effects, it is also possible to extend the life span of lotus tumor mouse.

Use the multiple different medicine carryings of similar Research on experimental methods (apoptotic proteins, toxic protein of plant, methotrexate, Therapeutic DNA and siRNA) crosslinking vesicle and targeting crosslinking the vesicle impact on tumor-bearing mice, result shows all can effectively press down Tumor processed increases, and mice is not had toxic and side effects, it is also possible to extend the life span of lotus tumor mouse.

Claims

1. inner membrance has a reversible crosslink Biodegradable polymer vesicles for positive electricity, is cross-linked by after polymer self assembles Arrive；The strand of described polymer includes hydrophilic segment, hydrophobic segment and the spermine molecule being sequentially connected with；Described hydrophobic segment Including Merlon segment and/or polyester segment；The molecular weight of described hydrophilic segment is 2000-8000Da；Dividing of hydrophobic segment Son amount is 2.3-8.4 times of hydrophilic segment molecular weight.

The most according to claim 1, inner membrance has the reversible crosslink Biodegradable polymer vesicles of positive electricity, it is characterised in that: The chemical structural formula of described polymer is as follows:

Wherein, R₁One in following group:

R₂One in following group:

、；

The most according to claim 2, inner membrance has the reversible crosslink Biodegradable polymer vesicles of positive electricity, it is characterised in that: The molecular weight of PEG is 4000-8000Da；The total molecular weight of PTMC or PLA is 2.5-5 times of PEG molecular weight；Total molecule of PDTC Amount is the 18%-38% of PTMC or PLA total molecular weight.

4. any one inner membrance described in claim 1-3 has the preparation of reversible crosslink Biodegradable polymer vesicles of positive electricity Method, it is characterised in that comprise the following steps:

(1) terminal hydroxy group of PEG-P (TMC-DTC) or PEG-P (LA-DTC) is activated by hydroxy activating reagent, more anti-with spermine PEG-P (TMC-DTC)-SP or PEG-P (LA-DTC)-SP should be prepared；

(2) at the PEG end coupling tumour-specific targeted molecular of PEG-P (TMC-DTC)-SP or PEG-P (LA-DTC)-SP, Obtain targeting PEG-P (TMC-DTC)-SP or targeting PEG-P (LA-DTC)-SP；

(3) with PEG-P (TMC-DTC)-SP or PEG-P (LA-DTC)-SP as raw material, prepare inner membrance by solvent displacement and have There is the reversible crosslink Biodegradable polymer vesicles of positive electricity；Or with PEG-P (TMC-DTC)-SP and targeting PEG-P (TMC- DTC)-SP is raw material, prepares inner membrance by solvent displacement and has the reversible crosslink Biodegradable polymer vesicles of positive electricity；Or Person as raw material with PEG-P (LA-DTC)-SP and targeting PEG-P (LA-DTC)-SP, prepares inner membrance by solvent displacement and just has The reversible crosslink Biodegradable polymer vesicles of electricity；Or with PEG-P (TMC-DTC)-SP and targeting PEG-P (TMC-DTC) For raw material, prepare inner membrance by solvent displacement and there is the reversible crosslink Biodegradable polymer vesicles of positive electricity；Or with PEG-P (LA-DTC)-SP and targeting PEG-P (TMC-DTC) is raw material, by solvent displacement prepare that inner membrance has positive electricity can Inverse crosslinked bio degradable polymer vesicle.

The most according to claim 4, inner membrance has the preparation method of reversible crosslink Biodegradable polymer vesicles of positive electricity, It is characterized in that, step (1) is at dry solvent by PEG-P (TMC-DTC) or PEG-P (LA-DTC), hydroxy activating reagent Middle reaction, then precipitates, filters, is vacuum dried the PEG-P (TMC-DTC) or PEG-P (LA-DTC) obtaining terminal hydroxy group activation； The PEG-P (TMC-DTC) activated by terminal hydroxy group or the solution of PEG-P (LA-DTC) are added drop-wise in solution of spermine reaction, then Dialysis, precipitation, sucking filtration, vacuum drying obtain PEG-P (TMC-DTC)-SP or PEG-P (LA-DTC)-SP；Step (2) is will PEG-P (TMC-DTC)-SP or PEG-P (the LA-DTC)-SP of step (1) and the targeted molecular being dissolved in organic solvent react To targeting PEG-P (TMC-DTC)-SP or targeting PEG-P (LA-DTC)-SP；Step (3) for by material solution adds non-from In sub-buffer solution, room temperature is dialysed after placing, is cross-linked, and obtains inner membrance and has the reversible crosslink biodegradable polymer capsule of positive electricity Bubble.

6. an antitumor drug, is had the reversible crosslinked polymer of positive charge by any one inner membrance described in claim 1-3 Vesicle loads medicine and obtains；Described medicine is the electronegative small-molecule drug of protein, nucleic acid or physiological environment.

7. the preparation method of antitumor drug described in claim 6, it is characterised in that the one in following preparation method:

(1) by PEG-P (TMC-DTC)-SP solution or PEG-P (LA-DTC)-SP solution and drug solution, nonionic buffer Mixing, room temperature is placed, and then dialyses, hatches crosslinking and obtain antitumor drug；

(3) PEG-P (LA-DTC)-SP solution and targeting PEG-P (LA-DTC)-SP solution are buffered with drug solution, nonionic Liquid mixes, and room temperature is placed, and then dialyses, hatches crosslinking and obtain antitumor drug；

(4) by PEG-P (TMC-DTC)-SP solution and targeting PEG-P (TMC-DTC) solution and drug solution, nonionic buffer Mixing, room temperature is placed, and then dialyses, hatches crosslinking and obtain antitumor drug；

(5) PEG-P (LA-DTC)-SP solution and targeting PEG-P (LA-DTC) solution are mixed with drug solution, nonionic buffer Closing, room temperature is placed, and then dialyses, hatches crosslinking and obtain antitumor drug.

8. any one inner membrance described in claim 1-3 has the reversible crosslink Biodegradable polymer vesicles of positive electricity as medicine The application of thing carrier；Described medicine is electronegative small-molecule drug under protein, nucleic acid or physiological environment.

9. any one inner membrance described in claim 1-3 has the reversible crosslink Biodegradable polymer vesicles of positive electricity in preparation Application in antitumor drug.

10. a polymer, it is characterised in that the chemical structural formula of described polymer is as follows:

Wherein, R₁One in following group:

R₂One in following group:

、；